1. Introduction Results Aim of the study Methods Conclusion References
Temocillin quantification in human serum using a high performance liquid chromatography-tandem mass spectrometry
P. Ngougni Pokem1,3, A.C. Miranda Bastos1,2,3, P.M. Tulkens1,3, F. Van Bambeke1,3, A. Capron4
Louvain Drug Research Institute: (1) Pharmacologie cellulaire et moléculaire, (2) Clinical Pharmacy Research Group, (3) Center for Clinical Pharmacy, Cliniques Universitaires Saint-Luc: (4) Department of Clinical Chemistry,
Université Catholique de Louvain, Brussels, Belgium.
Contact:
Introduction
Results
Temocillin (TMO) is a beta-lactam antibiotic that has recently seen both its usage and associated research increased, due to its remarkable resistance to beta-lactamases. [1]
Measuring TMO serum concentrations can be clinically useful for optimal patient management. A method using HPLC coupled to a UV detector has recently been developed and validated [2]. Yet, UV detection being not specific, interferences could occur when assaying TMO in the serum of patients taking multiple medications.
Linearity
Linearity of HPLC MS-MS detection of temocillin: Peak area ratio (TMO/IS) over the assay concentration range µg/ml
5 replicates of calibration standards over 3 days
Weight: 1/x R²
slope
Day 1
0.993
; P<0.001
Day 2
0.996
; P<0.001
Day 3
0.991
; P<0.001
Mean
; P<0.001
Standard deviation
0.0025
Coefficient of variation (CV%)
0.25
11.7
Method is linear
Aim of the study
To develop and validate a new HPLC method coupled to MS-MS detection for the analysis of temocillin in human serum.
Precision and Accuracy
TMO theoretical concentration
µg/mL
Mean estimated concentration
 intra-day (n=5)
inter-day (n=15)
Precision (CV%)
Accuracy (MRPE %)
Precision (CV%)
LLOQ 1
1.07
17.52
15.73
13.09
6.80
LQC 5
5.48
9.17
13.02
7.05
13.06
MQC 250
259.19
7.21
6.85
6.48
3.67
HQC 450
460.18
7.14
11.68
8.88
2.26
LLOQ: CV< 20%
Intra-day: CV < 15%
Inter- day: CV < 15%
Method is precise
Methods
LLOQ: MRPE<20%
Intra-day : |MRPE%| < 15%
Inter- day: |MRPE%| < 15%
Method is accurate
Methanol protein precipitation was used as the extraction method.
TMO calibration standards: 1 to 500 µg/mL.
TMO quality standards: 5 to 450µg/mL.
Internal standard: Ticarcillin (TIC) - final concentration 160µg/mL.
LLOQ, lower limit of quantification LQC, low quality control; MQC, medium quality control; HQC, high quality control
CV, coefficient of variation; MRPE, Mean relative prediction error
Sample preparation
200µL(serum + TMO) + 32µL TIC(1mg/mL) + 600µL methanol
Vortex for 5 sec
Centrifuge 11000g for 5 min
Remove supernatant to a vial
Recovery and Precision
TMO theoretical concentration
µg/mL
Mean % recovery
(n=6)
Standard deviation
Coefficient of variation (CV%)
LQC 5
85.80
6.90
8.04
LQC 25
89.15
10.63
11.92
MQC 250
90.96
6.86
7.54
HQC 450
99.40
7.50
7.55
Recovery is high, precise, reproducible, and is minimally affected by concentration
Equipment
HPLC (Alliance 2796 Waters)
MS-MS
Column
Xbrigde phenyl
(50 x 2,1mm; 3,5µm)
Ionisation mode
ESi+
Mobile phases
A: H2O+ 0,1%HCOOH
B: ACN + 0,1% HCOOH
Acquisition mode
MRM
Temperature
40°C
Transition (Da)
> (TMO)
> (TIC)
Flow rate
0,3 mL/min
Cone voltage (V)
20 (TMO)
25 (TIC)
Injection Volume
10µL
Collision Energies (eV)
12 (TMO)
14 (TIC)
Run time
6min
. comparison of analytical results for extracted samples at 3 concentrations (low, medium, and high ) with unextracted standards
. LQC, low quality control; MQC, medium quality control ; HQC, high quality control
Carry-over
Peak areas of TMO
500 µg/mL
Peak areas of blank sample (methanol)
Peak areas of LLOQ (1µg/mL)
Carry-over (%)
(n=5)
60.69
907.25
0.017
37.79
327.17
0.014
31.86
296.91
0.012
34.09
233.98
32.12
251.28
Carry-over < 0.1 % of LLOQ
Carry-over is in the acceptable range
Method validation according FDA acceptance criteria.
Chromatogram of a representative human serum sample at the LOQ
Gradient elution
Temocillin
Ticarcillin
. 5 successive injections of blank samples after the highest calibrator concentration.
Conclusion
References
This is the first report describing the quantification of temocillin by HPLC-MS/MS. This method proved fast, specific, and sensitive enough for determining temocillin levels in serum. It could be used for both pharmacokinetic studies and therapeutic monitoring purposes and should avoid any interference with other medications taken by the patients.
[1] Livermore DM, Tulkens PM. J Antimicrob Chemother (2009) 63:
[2] AC Miranda Bastos et al. J Pharm Biomed Anal. (2014) 90:
[3] Guidance for Industry: Bioanalytical Method Validation, U.S. Department ofHealth and Human Services, Food and Drug Administration, Center for DrugEvaluation and Research, 2001.
This poster will be made available for download after the meeting at